Mutated e. coli enterotoxins as anti-inflammatory agents

ABSTRACT

The invention provides compositions and methods for reducing symptoms of inflammation by administering therapeutically effective amounts of (a) (1) enterotoxic E. coli heat labile detoxified toxin A subunit that interferes with the function of ADP-ribosylation factor and inhibits ADP-ribosylation, or (2) A1 subunit that interferes with the function of ADP-ribosylation factor and inhibits ADP-ribosylation, or both (1) and (2), and (b) a carrier that causes internalization of the A subunit or A1 subunit, or both, into cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.Provisional Patent Application No. 62/667,992, filed May 7, 2018, thecontents of which are incorporated herein by reference for all purposes.

STATEMENT OF FEDERAL FUNDING

Not applicable.

BACKGROUND OF THE INVENTION

Inflammation may be thought of as the immune system's response to anirritant. Inflammation can promote fighting an infection or healing froma wound, and is helpful in those situations. Unfortunately, in someconditions, the body reacts unnecessarily and inflammation createsequally unnecessary discomfort. Diseases such as dermatitis or psoriasiscan result in inflammation of portions of the skin. Individual organs ororgan systems may have conditions, such as nephritis or asthma, whichare exacerbated by inflammation and for which treatment can includecorticosteroids or other anti-inflammatory drugs.

As the name implies, inflammatory bowel disease (“IBD”), a group ofinflammatory conditions of the gastrointestinal tract, most commonly inthe colon and small intestine, are diseases with strong inflammatorycomponents. Crohn's disease (“CD”) and Ulcerative Colitis (“UC”) are themajor types of IBD. These diseases are chronic inflammatory conditionsof the bowel and cause symptoms such as abdominal pain, diarrhea,vomiting, rectal bleeding, and can cause anemia. Currently, there are nocures for IBD but medication can be prescribed to manage the symptomsand improve quality of life for those with the disease. If symptoms ofIBD are not managed, abscesses or fistulae may form in severe cases,necessitating surgical removal of parts of the colon or small intestine.Surgical intervention can significantly impact the patients' quality oflife, potentially requiring the use of a colostomy bag.

There are a variety of treatments available for IBD. For mild cases,Non-Steroidal Anti-inflammatory Drugs (NSAIDs) may be prescribed;research has shown, however, that NSAIDs may have an adverse effect onIBD patients, sometimes worsening symptoms by damaging the lining of theintestines. Acetaminophen is another option but lacks effectiveness,except in mild cases.

Aminosalicylates are a family of anti-inflammatory compounds also usedto treat IBD. This class of drug is effective in mild to moderate casesof IBD, but may still have side effects such as pancreatitis, headache,vomiting, fever, stomach cramping, and diarrhea.

Steroidal treatments are another class of medication used for IBD thatare most useful during IBD flare ups due to their fast actinganti-inflammatory properties. However, these treatments are lesseffective at disease maintenance and can have significant side-effectsfrom long term use, including weakening the immune system.

Immunomodulators are another class of treatment for IBD. Thesemedications are effective against IBD but cause flu like symptoms andalso have the side effect of weakening the patients' immune systemreducing their ability to fight infection or other disease. Finally, afew biological treatments, including PDE inhibitors and monoclonalantibodies, such as Infliximab, have been used as a treatment for IBD.These biologics also have risk of side effects, including increasedinfections, serum sickness, and longer term side effects of decreasedeffectiveness of the drug, multiple sclerosis, and lymphoma.

Due to these potential short and long term side effects of managingthese chronic conditions, there is a need for a treatment for IBD thatprovides limited short and long term side effects, is highly effective,and has a strong safety profile. Further, it would be desirable to havenew agents to reduce symptoms of inflammation in other inflammatorydiseases, such as asthma, nephritis, dermatitis, and psoriasis.Surprisingly, the present invention fills these and other needs.

SUMMARY OF THE INVENTION

In a first group of embodiments, the invention provides compositionscomprising a unit dose of: (a) (i) an E. coli heat labile enterotoxin(“LT”) non-toxic A subunit which inhibits ADP-ribosylation in a cellpretreated with said non-toxic A subunit when said cell is thencontacted with E. coli. LT holotoxin, or (ii) a LT non-toxic A1 subunitwhich inhibits ADP-ribosylation in a cell pretreated with said non-toxicA subunit when said cell is then contacted with E. coli LT holotoxin,or, (iii) a combination of said non-toxic A subunit and of saidnon-toxic A1 subunit, and (b) a carrier which causes internalization ofsaid non-toxic A subunit or non-toxic A1 subunit, or combinationthereof, into cells. In some embodiments, the non-toxic A subunit or thenon-toxic A1 subunit, or the combination of the non-toxic A subunit andthe non-toxic A1 subunit does not induce intracellular cAMP accumulationin an epithelial cell. In some embodiments, the non-toxic A subunit orthe non-toxic A1 subunit, or the combination of the non-toxic A subunitand the non-toxic A1 subunit activates expression and secretion of IL-6in murine dendritic cells contacted with the non-toxic A subunit or thenon-toxic A1 subunit, or the combination of the non-toxic A subunit andthe non-toxic A1 subunit in vitro in a medium that supports growth ofsaid murine dendritic cells than the said cells when contacted with LT Bsubunit. In some embodiments, the carrier which causes internalizationof the non-toxic A subunit, non-toxic A1 subunit, or combinationthereof, into cells is a LT B subunit and the unit dose is from 0.5 mgor more of non-toxic A subunit, non-toxic A1 subunit, or combinationthereof and the B subunit carrier. In some embodiments, the carrierwhich causes internalization of the non-toxic A subunit, non-toxic A1subunit, or combination thereof, into cells is cholera toxin B subunit(“CTB”) or cholera A2 subunit/B subunit (“CTA2/B”). In some embodiments,the non-toxic A subunit, non-toxic A1 subunit, or combination thereof ischemically conjugated or recombinantly fused to the LT B subunit. Insome embodiments, the non-toxic A subunit, non-toxic A1 subunit, orcombination thereof is chemically conjugated or recombinantly fused tothe CTB or CTA2/B subunit. In some embodiments, the carrier which causesinternalization of the non-toxic A subunit, non-toxic A1 subunit, orcombination thereof, into cells is a liposome or encapsulating vesicle.In some embodiments, the liposome or encapsulating vesicle is aliposome. In some embodiments, the liposome has an exterior surface andhas an antibody or antigen-binding fragment or derivative thereofdisposed on the exterior surface. In some embodiments, the antibody orantigen-binding fragment or derivative binds CD11c. In some embodiments,the carrier which causes internalization of the non-toxic A subunit,non-toxic A1 subunit, or combination thereof, into cells is a β-glucan.In some embodiments, the unit dose composition does not contain anexogenous antigen. In some embodiments, the non-toxic A subunit,non-toxic A1 subunit, or combination thereof, is non-toxic A subunit. Insome embodiments, the non-toxic A subunit, non-toxic A1 subunit, orcombination thereof, is non-toxic A1 subunit. In some embodiments, thenon-toxic A subunit, non-toxic A1 subunit, or combination thereof, is acombination of non-toxic A subunit and non-toxic A1 subunit. In someembodiments, the composition is lyophilized. In some embodiments, thecomposition further comprises an excipient, a stabilizer, or both anexcipient and a stabilizer. In some embodiments, the non-toxic Asubunit, non-toxic A1 subunit, or combination thereof, has a mutation atposition E112, E110, S61, or R25. In some embodiments, the mutation isselected from E112K, E112G, E112D, E110K, E110G, S61F, and R25G. Insonic embodiments, the mutation is of E112K. In some embodiments, thecomposition is in a base suitable for topical administration. In someembodiments, the composition is in a liquid. In some embodiments, theliquid further comprises a flavoring agent and a sweetener. In someembodiments, the unit dose is from about 1 mg±0.2 to 500 mg of the Asubunit, A1 subunit, or combination thereof, and carrier. In someembodiments, the unit dose is from 1 mg±0.2 mg to 30 mg of the Asubunit, A1 subunit, or combination thereof and carrier. In someembodiments, the unit dose is from 1 mg±0.2 mg to 20 mg±0.2 mg of the Asubunit, A1 subunit, or combination thereof and carrier. In someembodiments, the composition is provided as a pill. In some embodiments,the composition is provided as a suppository.

In a further group of embodiments, the invention provides methods ofreducing symptoms of inflammation in a subject in need thereof. In someembodiments, the methods comprise administering to the subject acomposition comprising a therapeutically effective amount of: (a) (i) anE. coli heat labile enterotoxin (“LT”) non-toxic A subunit whichinhibits ADP-ribosylation in a cell pretreated with said non-toxic Asubunit when the cell is then contacted with E. coli LT holotoxin, (ii)a LT non-toxic A1 subunit which inhibits ADP-ribosylation in a cellpretreated with the non-toxic A subunit when the cell is then contactedwith E. coli LT holotoxin, or, (iii) a combination of the non-toxic Asubunit and the non-toxic A1 subunit, and, (b) a carrier which causesinternalization of the non-toxic A subunit or non-toxic A1 subunit, orcombination thereof, into cells. In some embodiments, the non-toxic Asubunit or non-toxic A1 subunit, or combination of non-toxic A subunitand non-toxic A1 subunit does not induce intracellular cAMP accumulationin an epithelial cell. In some embodiments, the non-toxic A subunit orthe non-toxic A1 subunit, or the combination of the non-toxic A subunitand the non-toxic A1 subunit does not activate expression and secretionof IL-6 in murine dendritic cells contacted with the non-toxic A subunitor the non-toxic A1 subunit, or the combination of said non-toxic Asubunit and the non-toxic A1 subunit in vitro in a medium that supportsgrowth of the murine dendritic cells, compared to the cells whencontacted with LT B subunit. In some embodiments, the carrier whichcauses internalization of said non-toxic A subunit, non-toxic A1subunit, or combination thereof, into cells is a LT B subunit, In someembodiments, the carrier which causes internalization of the non-toxic Asubunit, non-toxic A1 subunit, or combination thereof, into cells is acholera toxin B subunit (“CTB”) or cholera A2 domain and B subunit(“CTA2/B”). In some embodiments, the non-toxic A subunit, non-toxic A1subunit, or combination thereof is chemically conjugated orrecombinantly fused to said LT B subunit. In some embodiments, thenon-toxic A subunit, non-toxic A1 subunit, or combination thereof ischemically conjugated or recombinantly fused to the CTB or the CTA2/Bsubunit. In some embodiments, the carrier which causes internalizationof the non-toxic A subunit, non-toxic A1 subunit, or combinationthereof, into cells is a liposome or encapsulated vesicle. In someembodiments, the carrier is a liposome. In some embodiments, theliposome is targeted to a cell antigen by an antibody or antigen-bindingfragment or derivative thereof. In some embodiments, the carrier whichcauses internalization of the non-toxic A subunit, non-toxic A1 subunit,or combination thereof, into cells is a β-glucan. In some embodiments,the composition does not contain an exogenous antigen. In someembodiments, the non-toxic A subunit, non-toxic A1 subunit, orcombination thereof, is non-toxic A subunit. In some embodiments, thenon-toxic A subunit, non-toxic A1 subunit, or combination thereof, isnon-toxic A1 subunit. In some embodiments, the non-toxic A subunit,non-toxic A1 subunit, or combination thereof, is a combination ofnon-toxic A subunit and non-toxic A1 subunit. In some embodiments, thenon-toxic A subunit, non-toxic A1 subunit, or combination thereof, andthe carrier is lyophilized. In some embodiments, the composition furthercomprises an excipient. In some embodiments, the composition furthercomprises a stabilizer. In some embodiments, the non-toxic A subunit,non-toxic A1 subunit, or combination thereof has a mutation in said Asubunit, the A1 subunit, or both, selected from the group consisting ofE112K, E112G, E112D, E110K, E100G, S61F, or R25G. In some embodiments,the non-toxic A subunit, non-toxic A1 subunit, or combination thereofhas an E112K mutation in the A subunit, said A1 subunit, or both. Insome embodiments, the composition is administered in water. In someembodiments, the composition is administered orally. In someembodiments, the composition is administered rectally. In someembodiments, the composition is administered intra-nasally. In someembodiments, the composition is administered parenterally. In someembodiments, the parenteral administration is intravenous. In someembodiments, the composition is administered intramuscularly. In someembodiments, the composition is administered topically. In someembodiments, the composition is administered transdermally. In someembodiments, the therapeutically effective amount is administered as aninduction dose followed by one or more maintenance doses. In someembodiments, the composition is administered daily. In some embodiments,the therapeutically effective amount of said composition is from 500 μgto 500 mg. In some embodiments, the therapeutically effective amount ofsaid composition is from 80 μg to 500 mg. In some embodiments, thetherapeutically effective amount of said composition is from 1 mg to 250mg. In some embodiments, the therapeutically effective amount of saidcomposition is from 1 mg to 100 mg. In some embodiments, the subject inneed thereof is a mammal. In some embodiments, the mammal is selectedfrom a primate, feline, canine, bovine, equine, porcine, or ovine. Insome embodiments, the primate is a human. In some embodiments, theinflammation is gastrointestinal. In some embodiments, thegastrointestinal inflammation is inflammatory bowel disease. In someembodiments, the inflammatory bowel disease is ulcerative colitis. Insome embodiments, the inflammatory bowel disease is Crohn's disease. Insome embodiments, the inflammation is of the skin. In some embodiments,the inflammation of the skin is psoriasis or dermatitis. In someembodiments, the inflammation is a form of inflammatory arthritis. Insome embodiments, the inflammatory arthritis is rheumatoid arthritis,psoriatic arthritis, ankylosing spondylitis, or juvenile idiopathicarthritis. In some embodiments, the inflammation is of an internal organother than the small or large intestine, bowel, or colon. In someembodiments, the internal organ is a kidney, pancreas, or liver.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. FIG. 1A. FIG. 1A presents a ribbon structure diagram ofthe E112K non-toxic form of enterotoxic E. coli heat-labile toxin. Thediagram shows the A subunit disposed above the pentameric structureformed by five B subunits. The A1 subunit is connected to the pentamericstructure of the B subunits by the A2 domain of the A subunit, which isthe ribbon proceeding in a right-to-left diagonal from the top structureto the bottom structure. FIG. 1B. FIG. 1B is a photo of a SDS-PAGE gelof the A subunit, A1 domain, B subunit, and B pentamer of the E112Knon-toxic form.

FIGS. 2A-C. FIG. 2A. FIG. 2A presents brightfield images of confluent6-well plates of epithelial cells or macrophages that were cultured withmedia alone or combined with E112K doses for 24 hours in 2 mls totalvolume. Top panel: wells cultured with media alone. Bottom photographs:wells cultivated with E112K mutant. Left side, top and bottom: cellscultured in designated media were T84 epithelial cells. Right side, topand bottom: cells cultured in designated media were macrophages. FIG.2B. FIG. 2B presents graphs showing viability staining results of cellscultured with increasing amounts of E112K mutant, post-imaging bytrypsinizing cells, washing, and staining for cytometric analyses incomparison with cells killed by heating to 95° C. for 3 mn. Data isshown as % dead cells. Left graph: T84 epithelial cells. Right side:macrophages, FIG. 2C. FIG. 2C is a graph presenting the result of astudy of BALB/c mice administered the E112K mutant or different amountsof E. coli heat-labile toxin (“LT”) by oral gavage. After 3 hours, themice were euthanized and the gut/carcass weight ratios established. Theresults are shown in the graph. “TEAN” is a Tris buffer. (Significanceby one-way ANOVA analysis with Tukey's multiple comparison post-hoc testis shown as *P≤0.05, **P≤0.01 and ***P≤0.001 versus controls.)

FIGS. 3A-E. FIG. 3A. Caco-2 cells cultured in 12-well plates werestimulated with E. coli heat-labile toxin (“LT”), B subunit of LT(“LTB”) or E112K. After 1 day (˜18 h), 3, 5, or 7 days, cells werewashed extensively with phosphate buffered saline (“PBS”) and lysed withRIPA buffer containing protease inhibitor. FIG. 3A shows detection byWestern blot analysis of intracellular A- and B-subunits of lysates withanti-LTA and anti-LTB rabbit polyclonal sera. FIG. 3B. FIG. 3B is agraph showing intracellular cAMP pmol/ml levels in Caco-2 cells after 24h treatment with 1 or 0.1 μg of LT, E112K, or LTB. Caco-2 cells weregrown on 12-well transwell plates to confluency as measured by shortcircuit current (e.g., polarized epithelial cells). Cells werestimulated with LT, LTB, E112K or media. FIG. 3C. FIG. 3C is a graphshowing IL-6 secretion detected in apical compartment supernatant aftertreatment with lug protein treatment, only observed with LT. FIG. 3D.FIG. 3D presents two graphs. The left graph in 3D shows ion secretion (%change in current), and the right graph shows monolayer integrity (%change in resistance), 1.5, 3 or 4.5 h after 0.1 μg toxin treatmentmeasured by TEER assay. A negative current is an indicator of cellularpermeability. FIG. 3E. Caco-2 cells were treated with 0.1 μgtrypsin-activated LT with or without a 10 min pre-treatment with 1 μgE112K, prior to 3 h incubation and intracellular cAMP analysis. Theresults are presented in FIG. 3E. (Significance by one-way ANOVAanalysis with Tukey's multiple comparison post-hoc test is shown as***P≤0.001 versus naïve control.)

FIGS. 4A-D. FIG. 4A. 1×10⁶ bone-marrow derived dendritic cells werecultured in a 24-well plate stimulated with LT, LTB or E112K. After 18h, dendritic cells were washed extensively with PBS and lysed with RIPAbuffer containing protease inhibitor. Detection of intracellular A- andB-subunits was performed by Western blot analysis using whole celllysates (WL), or cytosol, membrane and cytoskeleton cellular fractions(C, M, SK, respectively). FIG. 4A presents photographs of the Westernblots. FIG. 4B. 1×10⁶ bone-marrow derived dendritic cells were treatedwith 0.1 μg LT and/or E112K for 24 h in the presence ofphosphodiesterase inhibitor prior to cell lysis and cAMP assay. FIG. 4Bis a graph presenting the results. Y axis: intracellular cAMP (pmol/ml).X axis: cells treated with LT, with E112K, or untreated, respectively.FIG. 4C. FIG. 4C presents a photograph of a Western blot. 1×10⁶bone-marrow-derived dendritic cells were left untreated (lane 1),treated with 1 μg LT for 3 h (lane 2) or pre-treated with 1 μg E112K for24 h before 3 h 1 μg LT treatment (lane 3). Detection of ADP-ribosylatedprotein in whole cell lysates by Western blot was performed usingmacrodomains. FIG. 4D. FIG. 4D is a graph presenting the results of astudy showing 1×10⁶ bone-marrow derived dendritic cells activation after48 hours of LT or LPS treatment with or without a 3 hour pretreatmentwith 0.5 μg/ml E112K or LTB. Activation was assessed by surface stainingand analysis of dendritic cell (CD11c) and co-stimulatory (CD80) markersby flow cytometry

FIG. 5. FIG. 5 is a graph presenting the results of a study in whichBALB/c mice were immunized intradermally with 50 μl containing 10 μgtetanus toxoid and 0.1-5 μg LT, LTB, E112K or no adjuvant (designated ongraph as “none”). After 21 days, serum was collected and analyzed byELISA for presence of anti-tetanus toxoid antibodies.

FIGS. 6A-B. Both figures: The classic dextran sulphate sodium (“DSS”)chemical injury model of chronic, ulcerative colitis was induced bysuccessive waves of 5-day drinking water treatment with 4% DSS, followedby 7-days of water in BALB/c mice. After the 2nd (day 17) or 3rd (day29) DSS treatment, some mice were treated with 50 μg E112K by oralgavage. FIG. 6A. FIG. 6A presents two graphs. The top graph shows finalweight change (day 36, % original weight), while the bottom graph showscolon length (day 38, mm) as evaluated for all groups. FIG. 6B. FIG. 6Bpresents H&E photographs of swiss-rolled, processed colonic mouseintestines (from day 38 collected samples). Top photograph: watertreatment. Middle: DSS treatment. Bottom: DSS treatment+E112K therapy.The middle photograph shows DSS-mediated cellular infiltration (see areaaround black asterisk), while a moderation of DSS-mediated cellularinfiltration is seen with E112K therapy (bottom photograph).

FIGS. 7A-D. FIGS. 7A-D present a study of inflammatory bowel diseasepathology in IL-10-/- colitis model. FIG. 7A. FIG. 7A is a graph showingthat weight gain over time after 200 μg E112K by oral gavage in IL-10-/-female and male mice are similar to that of naïve mice. FIG. 7B. Thechronic colitis model was induced in IL-10-/- mice with 7 day exposureto 200 ppm piroxicam at 6-weeks of age. Piroxicam diet (in food gel,formulated food, or powder food) induces colitis pathology at week 10.Colon histology scores were tallied using composite scoring of 4 colonicsections for type and extent of mucosal and inflammatory changes bypathologist blinded to animal treatments. As shown in FIG. 7B, 200 μgE112K by oral gavage protected the mice from induction of intestinalpathology from the piroxicam diet. FIG. 7C. FIG. 7C presents aphotographic example of H&E-stained colons from piroxicam-treatedanimals exhibiting typical pathology including hyperplasia (top,asterisk) and ulceration of intestinal wall (bottom, asterisk), FIG. 7D.The chronic colitis model was induced in IL-10-/- mice with 7 dayexposure to 200 ppm piroxicam at 6-weeks of age followed by weekly E112Ktreatment by intraperitoneal injection or oral gavage for three weeks.FIG. 7D presents the results in a graph comparing the colon histologyscore of untreated animals (“untx”), animals treated with 1 μg or with10 μg by intraperitoneal injection (IP) or with 10 μg or with 100 μg byoral gavage (“O”). FIG. 7E. FIG. 7E is a graph presenting the results ofa study of intestinal permeability. Groups of mice (n=5-6) were exposedto piroxicam and then either untreated (“untx”) or treated by oralgavage of E112K weekly for three weeks (“100 μg O”). On week 10, micewere fed 4 kD FITC-Dextran at 60 mg/100 g body weight by oral gavage 3 hprior to euthanasia. Serum was collected and then analyzed for levels offluorescent protein after 3 h oral gavage with at week 10. Significanceis shown as *P≤0.05, **P≤0.01 and ***P≤0.001.

FIGS. 8A-D. FIGS. 8A-D show that E112K therapy improves colitis inT-Cell Transfer Colitis Model. FIG. 8A. FIG. 8A is a diagrammaticoverview of the T-cell transfer model of colitis. Rag1-/- mice received2.5e5 CD4+CD45RB-hi T-cells by intraperitoneal injection on week 7 andwere then exposed to piroxicam in food for 7 days at week 7 to induceintestinal inflammation. Some mice were left untreated, whereas othersreceived 100 μg E112K by oral gavage on weeks 13, 16, and 18 (O E112K).After week 20, animals were euthanized. FIG. 8B. FIG. 8B is a graphshowing the assessment of the euthanized animals for colitis by blindedpathology scoring. FIG. 8C. FIG. 8C is a graph showing the change inweight of the animals (as % weight change from week 7 weight). FIG. 8D.FIG. 8D is a graph comparing the stool consistency of the treated and ofthe untreated animals.

FIG. 9. FIG. 9 shows a photograph of a SDS-PAGE gel of purified E112Kstained with Coomasie Blue. Twenty μg of the proteins identified in thelegend on the left were loaded into the well identified for therespective proteins, including native LT (LTh), E112K (lot #16001), orother mutant proteins compared to 5 μg of SeeBlue Plus 2 standard. Someproteins were exposed to 10 ng trypsin for 1 h at 37C prior to gelanalysis.

FIG. 10. FIG. 10 presents two graphs showing that mutants like E112Khave minimal ability to stimulate cAMP in cultured T84 human colonicepithelial cells, in comparison with native LT. cAMP analyses wereperformed by testing treated cell lysates. Briefly, lyophilized lots ofE112K (indicated by the numbers in parentheses) were re-suspended andtested at the various doses indicated, using 24-well seeded T84epithelial cells pre-treated with phosphodiesterase inhibitors. After 3h, cells were harvested, washed, and lysed for analyses of intracellularcAMP using cAMP Parameter Assay Kit (R&D Systems) at 1:20 dilutions (topgraph) or 1:2 dilution (bottom graph).

FIG. 11. FIG. 11 is a graph showing that E112K improves intestinalpermeability as soon as 24 h post-treatment. The chronic colitis modelwas induced in IL-10-/- mice with 7 day exposure to 200 ppm piroxicam at6-weeks of age. IL-10-/- were exposed to piroxicam for 7 days in rodentchow and then 4 days later either left untreated or treated with E112Kin drinking water. Intestinal permeability was performed 1-day aftertreatment by analyzing serum for fluorescent protein after 3 h oralgavage with 4 kD FITC-Dextran. Significance is shown as *P≤0.05.

FIG. 12. FIG. 12 presents two graphs showing that E112K improves diseaseactivity index in DSS acute model of colitis in immunodeficient mice.Rag1-/- that lack functional adaptive immune system were exposed to 3%DSS in drinking water for 7 days. A group of these DSS mice were treatedwith oral E112K on day 5. Weight changes and disease activity index(evaluated by weight loss, fecal consistency, and blood in feces) wereevaluated in animals at day 8. E112K treatment improved disease activityindex, but not weight loss, indicating that at least some of E112Kimmunomodulatory effects are T-cell independent.

FIGS. 13A-C. FIGS. 13A-C presents graphs showing that LT-based AB5adjuvants (LT, dmLT, mLT) mature dendritic cells and promoteTh17-promoting cytokine secretion, while LTB and E112K act distinctly.FIG. 13A. 1×10⁶ bone-marrow derived mouse dendritic cells were treatedin triplicate for 48 h with 0.1 μg/ml test proteins before analysis ofCD80 and CD86 co-stimulatory marker levels on CD11c+ gated cells. Assaywas performed three independent times with representative dot blots andmean fluorescent intensity levels (MFI) from one experiment shown.Th2-promoting cytokines and Th1/Th17-promoting cytokines in culturesupernatants of 1×10⁶ CD11c+ purified DC cultured with 0.1 μg/ml testproteins in triplicate for 5 days. FIG. 13B. FIG. 13B is a graphpresenting the results for Th2-promoting cytokines. FIG. 13C. FIG. 13Cpresents six graphs. The top two graphs show the results for twoTh1-promoting cytokines, IL-12p70 and RANTES. The bottom four graphsshow the results for four Th17-promoting cytokines, IL-1β, IL-8, IL-6,and G-CSF, respectively. Legend: “mLT” is a mutated LT with the mutationR192G in the A subunit. “dmLT” is a double-mutated LT with the mutationsR192G and L211A in the A subunit.

FIG. 14. FIG. 14 presents a proposed mechanism of E112K suppression ofinflammation. Binding to surface receptors like GM1 on cells, includingepithelial cells, results in release of E112K A-subunit to bind andinhibit host cells ADP-ribosylation factor (ARF). This suppresses therelease of inflammatory cytokines, signaling responses, andinflammation.

DETAILED DESCRIPTION

Surprisingly, it has now been discovered that non-toxic A subunits ofentertoxic E. coli (“ETEC”) heat-labile toxin (“LT”) that interfere withthe function of host cell ADP-ribosylation factor (“ARF”) and inhibitADP-ribosylation also inhibit the activation of dendritic cells andreduce acute and chronic inflammation, including inflammatory T-cellactivity. In studies underlying the present disclosure, an exemplardetoxified A subunit and carrier that allowed it to be internalized intocells was administered to animals in several different models ofinflammatory bowel disease (“IBD”). Animals treated with the exemplarconstruct in the studies exhibited improvements in pathology scores,showed no abnormal loss of weight, and showed normal stool consistency.In view of these results, it is believed that this detoxified A subunit,and other detoxified A subunits that likewise interfere with ARTintracellular function and inhibit ADP-riboyslation will also inhibitthe activation of dendritic cells, reduce acute and chronicinflammation, including inflammatory T-cell activity, and improvesymptoms of IBD. Further, a study underlying the present disclosureshowed rapid improvement of barrier permeability in the intestine in amurine chronic colitis model, suggesting the constructs were taken up bymucosal epithelial cells and the tissue resident myeloid cells andmacrophages and ameliorated symptoms of colitis due to increasedpermeability. A further study in a murine model using immunodeficientmice showed that mice treated with an exemplar detoxified A subunit andcarrier showed improved fecal consistency and reduced blood in feces,but did not improve weight loss, indicating that some of the detoxifiedA subunit effects are not T-cell dependent. Finally, studies underlyingthe present disclosure revealed that non-toxic A subunits have asurprisingly different effect on expression and secretion ofinflammatory cytokines than do either the LT B subunit or versions ofthe ETEC holotoxin that act as adjuvants for antigens.

In view of these results, use of non-toxic A subunits with carriers thatcan cause uptake and internalization of the A subunits into dendriticcells in persons with IBD are expected to reduce and treat symptoms ofIBD, in particular, intestinal or bowel inflammation, diarrhea, andblood in stool. Improvement in any one of these symptoms, or anycombination of them, is expected to improve patient quality of life.Additionally, as activation of dendritic cells is a common feature ofinflammatory diseases, it is believed that non-toxic A subunits thatinhibit ADR-riboyslation provided in a carrier that allowsinternalization into cells will also reduce inflammation in conditionsother than IBD. Thus, the studies underlying the present disclosurerepresent an important advance both in treating IBD, and more generally,in treating inflammation in other conditions in which inflammationexacerbates the condition, such as asthma, dermatitis, psoriasis,psoriatic or rheumatoid arthritis, and nephritis.

As noted, the detoxified A subunits that inhibit ADR-riboyslation areprovided in a carrier that allows internalization into cells. Results ofstudies underlying the present disclosure indicated that the anti-IBD,anti-inflammatory results seen were due to the detoxified A subunit, notthe B subunit. Thus, it is believed that the same anti-IBD,anti-inflammatory effects shown in the studies would be seen if thedetoxified A subunit is delivered by other means, such as by using as acarrier the cholera toxin B subunit (“CTB”), cholera toxin A2 domain andCTB (“CTA2/B”), or encapsulating the detoxified A subunit in liposomesor other vesicles, or coupling the detoxified A subunit to othercarriers that induce internalization of the detoxified A subunit intocells.

In the studies underlying the present disclosure, the detoxified Asubunit (the terms “non-toxic” A subunit and “detoxified” A subunit areused as synonyms in this disclosure) was delivered to cells by theentertoxic E. coli (“ETEC”) LT B subunit. (More precisely, the carrierin these experiments was the pentameric structure formed by five Bsubunits. As the individual B subunits comprising the pentamericstructure are not used individually, but only as part of the pentamericstructure formed by the five subunits, for convenience of reference,unless specified otherwise, the term “B subunit” herein refers to thepentameric structure formed by five B subunits of the E. coli LT or tothe similar pentameric structure formed by five B subunits of thecholera toxin, where the discussion relates to the cholera toxin Bsubunit.)

In these studies, the detoxified A subunit comprised both the A1 domainof the A subunit, which is responsible in the native toxin for the toxiceffects, and the A2 domain of the A subunit, which serves tonon-covalently tether the A1 domain to the LT B subunit. The A2 domainis not needed if a carrier other than the ETEC LT B subunit is used,such as CTB, liposomes, and other encapsulated vesicles. Thus, at thepractitioner's choice, in those embodiments, the detoxified A subunitmay be just the A1 domain of the A subunit, may comprise the intact Asubunit, or may comprise some molecules of the A1 domain and some of theintact A subunit.

Because enterotoxic E. coli heat-labile toxin is clinically important asa cause of traveler's diarrhea, there has been some investigation ofETEC LT A subunits, in combination with the LT B subunit, as vaccinesagainst ETEC-related traveler's diarrhea, and for use as adjuvants.Detoxified A subunit mutants that inhibit ADP-ribosylation are useful asanti-inflammatory agents, but are either not useful as adjuvants toenhance a response to an antigen. With regard to vaccine formulations,one reported Phase 3 clinical trial tested LT holotoxin (intactwild-type toxin) as a vaccine administered transdermally via patch. Thepatch in the clinical trial contained 37.5 μg E. coli LT. Behrens etal., The Lancet, 2014, 14(3):197-204.

Studies in which cells were pretreated with the exemplar compositionE112K and then contacted with native holotoxin did not exhibitADP-ribosylation. The inference is that the E112K composition bindsADP-ribosylation factor (“ARF”), thereby interfering with or blockingits ADP-ribosylation activity.

As shown in FIG. 13, studies comparing E112K to native holotoxin, to LTB subunit, and to two mutated forms of holotoxin tested as adjuvants,found the effect of E112K on expression of activation markers andsecretion of proinflammatory cytokines to be surprisingly different thatthe effect of the holotoxin, of LT B subunit, or of either of the twomutated forms of holotoxin tested as adjuvants. In contrast toholoenzyme, to LT B subunit, and to the two mutated forms of theholotoxin, E112K did not induce production of either Th1-promotingcytokines assayed or of any of the four Th17-promoting cytokinesassayed. Accordingly, E112K was shown to have surprising, andsurprisingly strong, anti-inflammatory properties.

Finally, in animal models of inflammatory bowel disease underlying thepresent disclosure, animals administered E12K exhibited less blood instool, better fecal consistency and, in some studies, less weight lossthan did animals not treated with E112K. In some embodiments, a subjectwith an IBD and exhibiting any one of: less blood in stool, better fecalconsistency and less weight loss, when administered one of the inventivecompositions or treated according to one of the inventive methods isconsidered to exhibit reduced symptoms of inflammation from the IBD.

Definitions

The terms “exogenous antigen” in relation to a non-toxic LT A subunit ornon-toxic A1 subunit, or both, and a carrier, means that the compositionof (a) the A subunit or A1 subunit or both, and (b) the carrier does notalso carry with it as an additional component an antigen which willraise an immune response in the subject to which the composition isadministered.

The term “carrier” as used herein in relation to a compositioncomprising a non-toxic LT A subunit or non-toxic A1 subunit, or both,and a carrier, refers to a molecule which, when associated with, fusedto, or conjugated to the A subunit or to the non-toxic A1 subunit,facilitates entry of the A subunit or non-toxic A1 subunit into cells.The entry into the cell can be by any means of uptake, such asendocytosis, vesicle-mediated transport, an active transport pathway, orreceptor-mediated uptake, depending on the particular carrier employed.

The phrase “a non-toxic LT A subunit or non-toxic A1 subunit, or both”means a composition can comprise (a) one or more non-toxic A subunits,(b) one or more non-toxic A1 subunits, or (c) one or more non-toxic Asubunits and one or more non-toxic A1 subunits.

References to mutated A subunits or A1 subunits herein follow theconvention of naming the amino acid residue at a particular position inthe designated protein, the number of the position, and then the aminoacid residue substituted for the original residue at that position.Letters representing the various amino acid residues also followart-recognized conventions. Thus, for example, E112K describes thesubstitution of a lysine, K, for a glutamic acid, E, at position 112 ofthe LT A subunit.

The terms “effective amount” or “therapeutically effective amount” of acomposition, as provided herein, refer to a nontoxic but sufficientamount of the composition to provide the desired therapeutic effect, oran amount sufficient to effect treatment of the subject, as definedbelow. The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the condition being treated, and the particularmacromolecule of interest, mode of administration, and the like. Anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

The phrase “pharmaceutically acceptable,” in connection withadministration of a substance to a human refers to a substance that isgenerally safe for human pharmaceutical use. In connection withadministration to a non-human animal of a particular species, it refersto a substance that is generally safe and acceptable to a non-humananimal of the species in question.

As used herein, the terms “pharmaceutically acceptable carrier” and“pharmaceutically acceptable vehicle” are interchangeable and refer to afluid vehicle for containing enterotoxin anti-inflammatoriescompositions that can be injected into a host without adverse effects oradministered to a host by another route of administration withoutadverse effects, depending on the intended route of administration.Suitable pharmaceutically acceptable carriers known in the art include,but are not limited to, sterile water, saline, glucose, dextrose, orbuffered solutions. Carriers may include auxiliary agents including, butnot limited to, diluents, stabilizers (i.e., sugars and amino acids),preservatives, wetting agents, emulsifying agents, pH buffering agents,viscosity enhancing additives, colors and the like.

E. Coli Enterotoxins and Non-Toxic LT A Subunits and LT A1 Domain

According to the website of the Centers for Disease Control andPrevention: “Enterotoxigenic Escherichia coli (E. coli), or ETEC, is animportant cause of bacterial diarrheal illness. Infection with ETEC isthe leading cause of travelers' diarrhea and a major cause of diarrhealdisease in lower-income countries, especially among children.” Certainstrains of enterotoxigenic E. coli produce a heat-labile enterotoxin(“LT”). It has been established for many years that LT induces cAMPaccumulation through binding to host cell ADP-ribosylation factor(“ARF”) to initiate ADP-ribosylation of Gsα, leading to irreversibleactivation of adenylate cyclase and increased production ofintracellular cAMP, which ultimately leads to secretory diarrhea.

LT has an AB₅ structure with non-covalently attached A- and B-subunits.The A-subunit causes the enterotoxic effects of the holotoxin, while theB (“binding”) -subunit is responsible for cellular binding andinternalization. A ribbon diagram of a partially-inactivated form of theholotoxin (discussed below) is presented in FIG. 1A, which shows the Asubunit having a portion extending into a pentameric structure formed byfive subunits. The 28 kD A-subunit is susceptible to cleavage bytrypsin, resulting in a 21 kD A1 domain. The 56 kD B-subunit pentamer ismore proteolytically stable and only breaks down into 11 kD monomersupon boiling.

The nucleic acid and amino acid sequences of both the A and the Bsubunits have been known for decades, as exemplified by Yamamoto et al.,J. Bacterial 1987, 169:1352-57, which presents the nucleic acid andamino acid sequences of both the A and the B subunits for the form foundin human isolates (shown in the paper as “LTh”), as well as the nucleicacid and amino acid sequences of the cholera toxin A and B subunits.

E112K is derived from LT, with a single amino acid substitution of alysine residue in place of a glutamic acid residue at position 112 inthe A1 domain (FIG. 1A). The mutation disrupts the active site and theability of the molecule to ADP-ribosylate host receptor proteins. Tsujiet al, J Biol Chem, 1990, 36:22520-525. FIG. 6 of Tsuji et al. shows thenucleic acid and amino acid sequences of the entire A subunit, and noteswhich portions of the overall sequence constitute the A1 domain andwhich constitute the sequence of the A2 domain of the A subunit.

As shown in the studies reported in the Examples, below, E112K proteinis non-toxic and exhibits no alteration in cell viability of culturetreated macrophages or epithelial cells (FIGS. 2A, 2B). A classiccharacteristic of native LT is induction of fluid secretion duringenterotoxigenic Escherichia coli (“E. coli”), or “ETEC” infection orwith purified LT alone. However, the mutation in E112K preventsinduction of intestinal fluid secretion (FIG. 2C).

The E112K construct used in the Examples was the A subunit with an E112Kmutation, tethered through the A2 domain to an ETEC LT B subunit, whichacted as the carrier to allow internalization of the A subunit and the Bsubunit into cells, such as dendritic cells. References to “E112K” inthe Examples refer to this construct. References in other sections ofthe disclosure refer to this construct when referring to the Examples,but in other places refer either to this construct or to an A subunit orA1 subunit, which may be paired either with an ETEC LT B subunit, orwith a different carrier, such as a cholera toxin B subunit (“CTB”), A2domain/B subunit (“CTA2/B”), a liposome, and other carriers describedelsewhere in this disclosure.

E112K has (1) no ability to ADP-ribosylate host receptor proteins, (2)no ability to induce intracellular cAMP accumulation in epithelialcells, and (3) no ability to act as a robust adjuvant forco-administered antigens. Further, as shown in the Examples and in FIG.13, studies underlying the present disclosure showed that E112K had asurprisingly different effect on the expression of activation markersand secretion of pro-inflammatory cytokines than did either the Bsubunit or of mutated forms of the A subunit that have been shown to beable to adjuvant responses to antigens. In particular, as shown in FIG.13, mouse dendritic cells contacted with E112K in vitro in a mediumsupporting dendritic cell growth, showed do not express and secrete IL-6or other pro-inflammatory cytokines. For purposes of this disclosure, a“non-toxic A subunit” or a “non-toxic A1 domain” means an A subunit orA1 domain which, when delivered to cells, shares these properties.

Several other mutated forms of the A1 domain are believed to share thesefunctional properties. These forms are: E112G, E112D, E110K, E110G,E110D, S61F, and R25G. Given these shared functional properties, it isbelieved that each of these other mutated forms of the LT A subunit andof the A1 domain are non-toxic and will also be unable to induceinflammatory cytokines when internalized into a cell. It is thereforebelieved that each can be used as anti-inflammatory agents. Since it isthe A1 domain of the LT A subunit that is responsible for the toxiceffect of LT (as noted, the A2 domain serves as a tether to the Bsubunit, and is not believed to participate in interference with ARFfunction or inhibition of ADP-ribosylation), references to mutations inthe A1 domain or subunit also refer to mutations in the A subunitcomprising both the A1 domain and the A2 domain. Further, forconvenience of reference, reference herein to “A1 subunit” whenreferring to the E. coli LT means the A1 domain of the LT A subunit, orof mutated forms of the LT A1 domain of the A subunit, as required bycontext.

While the eight mutants described above are exemplary of mutations inthe A1 subunit that remove the ability of the A1 subunit to inducediarrhea and to avoid other adverse effects, they are only some of thevariations in the A subunit or A1 subunit that are expected to havethese effects. For example, in E112K, the acidic amino acid residue E,glutamic acid, which is negatively charged at physiological pH (allreferences to charge in this section refer to charge while in an aqueoussolution at physiologic pH), is replaced with a K, a basic amino acidthat is positively charged, while in E112D, the glutamic acid has beenreplaced with D, aspartic acid, another negatively charged residue. Thissuggests that E112, which is at the active site of the A1 subunit, isparticularly sensitive to mutations and that the toxic properties of A1would also be abrogated at least by substitution of the E with anotherpositively charged amino acid, arginine (“R”). Similarly, in S61F, theuncharged, polar serine (“S”) residue at position 61 is replaced with F,phenylalanine, an amino acid with a hydrophobic side chain terminatingin a phenyl ring, suggesting that substituting the S with a tyrosine(“Y”) residue, whose side chain also terminates in a phenyl ring, butwith a hydroxyl attached to the ring, will also result in inactivationof the A1 subunit's enzymatic properties. In the R25G detoxified A1subunit, the arginine, R has been substituted by a small, unchargedamino acid, G, glycine, suggesting that substituting the positivelycharged R with either of the negatively charged residues E or D willalso result in inactivation of the A1 subunit. Other substitutions willreadily suggest themselves to persons of skill in the art and canreadily be tested to see if they result in (1) inhibition ofADP-ribosylation of host receptor proteins, (2) no ability to inducecAMP in epithelial cells, and (3) no ability to act as a robust adjuvantfor co-administered antigens. In some embodiments, therefore theinventive compositions and methods contemplate use of detoxified formsof the A1 subunit sharing these three properties. Other mutations ofresidues at the active site of the A subunit's ADP-ribosylation activitythat confer these properties are also comprehended.

In some embodiments, the detoxified A1 subunit is E112K. In someembodiments, the detoxified A1 subunit is E112G. in some embodiments,the detoxified A1 subunit is E112D. In some embodiments, the detoxifiedA1 subunit is E110K. In some embodiments, the detoxified A1 subunit isE110G. In some embodiments, the detoxified A1 subunit is E110D. In someembodiments, the detoxified A1 subunit is S61F. In some embodiments, thedetoxified A1 subunit is R25G.

Delivery of a Subunit by Means Other Than the LT B-Subunit

The B-subunit mediates binding of the holotoxin to cells andinternalization into them. The B-subunit binds to ganglioside receptors,such as GM₁ (monosialotetrahexosylganglioside) on gut epithelial cellmembranes and dendritic cells. There are several types of B-subunits,Type I and II A and B, which differ in amino acid sequence and theextent to which they bind different ganglioside receptors. See, e.g.,Tinker et al., Infection and Immunity, 2005, 73(6): 3627-55. It isbelieved that the anti-inflammatory effects seen in the studiesunderlying the present disclosure are due to the detoxified A1 subunitand not to the B subunit. Thus, it is believed that the potentanti-inflammatory effects shown in the studies reported in the Examplescan be achieved by delivering a detoxified A1 subunit to dendritic cellsand other cells of interest using means of delivery other than an LT Bsubunit.

In some embodiments, it is contemplated delivering a detoxified A1subunit to cells of interest using the pentameric cholera toxin Bsubunit (“CTB”) or the non-toxic cholera toxin CTA2/B.

Chimeras have been made and have demonstrated the efficacy of CTB as acarrier for antigens for some thirty five years, as exemplified by,e.g., McKenzie and Halsey, J Immunol, 1984, 133 (4) 1818-1824(horseradish peroxidase (HRP) covalently attached to CTB was shown toraise order of magnitude greater amounts of anti-HRP antibody than HRPor CTB alone), In 1990, a genetic construct of nucleic acid encodingglycosltransferase at the N-terminal of CTB was made and shown tomaintain structure and function of CTB. The authors stated the study“demonstrated a complete system for constructing, expressing, andpurifying cm chimeras.” Dertzbaugh et al., Infect. Immun. 1990,58(1):70-79, at p. 78. By 2001, researchers in the area were able tostate “It is well established that CTB is a highly efficient carriermolecule for the induction of mucosal antibody responses . . . as wellas for the induction of mucosally induced systemic T-cell . . . andsystemic B-cell . . . tolerance.” George-Chandy, et al., Infect Immun.2001, 69(9):5716-25 (“George-Chandy”), at p. 5723. (Citations omitted.George-Chandy reported in their study that antigen chemically conjugatedor genetically fused to CTB “dramatically lowers the thresholdconcentration of antigen required for immune cell activation.” See,Abstract.)

Both CTB and constructs of the A2 domain of cholera toxin (“CTA2”) incombination with CTB (“CTA2/B”), have been shown to deliver exogenousproteins to cells. See, e.g., Li et al., Infect. Immun. 2004,72:7306-7310; Tinker et al., Toxins 2014, 6(4), 1397-1418 (West NileVirus DIII-CTA2/B chimera shown to be immunogenic after intranasaldelivery). CTB has been used to deliver antigens orally and to serve asan adjuvant for mucosal delivery of antigens intranasally, rectally, andvaginally. See, e.g., Holmgren, et al., Vaccine, 1993, 11(12):1179-84;Hajishengallis et al., J Immunol, 1995, 154(9):4322-4332; Langridge etal., Current Opin Investig Drugs, 2010, 11(8):919-928. George-Chandy,supra, reported that chemically conjugating antigen to CTB or expressingthe antigen and CTB as a fusion protein resulted uptake of antigen intocells through the GM1 ganglioside receptor, showing that proteinsconjugated or fused to the CTB are taken into the cell.

In short, reports over the past three decades have shown that work hasshown that a variety of proteins have been successfully recombinantlyfused or chemically conjugated to CTB and CTA2/B and successfullydelivered into target cells both in vitro and in vivo. It is thereforeexpected that non-toxic LT A subunits that bind to ARF can begenetically fused or chemically conjugated to CTB or to CTA2/B by themethods developed over the past three decades. It is further expectedthat such fusions or conjugates will be delivered into cells just likethe numerous fusions and constructs already demonstrated to deliverproteins into cells, as exemplified by the antibodies that have beenraised against the proteins. Since the LT A2 subunit serves as a linkerto the B subunit, but does not participate in interaction with ARF, itis expected that the A2 portion of the A subunit can be omitted in suchchimeras, reducing the size of the LT protein to be fused or conjugatedto CTB or CTA2/B.

In some embodiments, the detoxified A subunit can be loaded intoliposomes or other encapsulating vesicles. Loading therapeutic agentsinto liposomes has been known since the 1980s. See, e.g., Woodle andStorm, eds., LONG CIRCULATING LIPOSOMES Old Drugs, New Therapeutics,(Springer-Verlag Berlin Heidelberg, 1998), Lasic and Papahadjopoulos,eds., MEDICAL APPLICATIONS OF LIPOSOMES (Elsevier Science B.V.,Amsterdam, 1998), Gregoriadis, G., ed., LIPOSOME TECHNOLOGY 2^(ND)EDITION, ENTRAPMENT OF DRUGS AND OTHER MATERIALS, vols, I and II (CRCPress, Inc., Boca Raton, Fla., 1993). Liposomes or other encapsulatingvesicles known in the art are typically taken up by myeloid cells andendocytosed, delivering the inventive compositions into cells of theimmune system that can initate reduction of inflammatory symptoms.

In some embodiments, the liposomes or other encapsulating vesicles canbe targeted to cell types of interest by tethering antibodies orfragments (e.g., Fab, F(ab′)2) or variable region fusion proteins (e.g.,single chain variable fragments, or “scFv”) of antibodies that bindantigen to the exterior of the liposomes. Methods of targeting liposomesto target cells by tethered antibodies, antigen-binding portions, orfusion protein derivatives thereof, such as Fab, F(ab′)2, and scFvs,have been known since at least the early 2000s, as exemplified by, e.g.,U.S. Pat. Nos. 6,210,707 and 6,214,388. In some embodiments, theantibodies or antigen-binding fragments or single chain variablefragments thereof bind CD11c. According to Martin, A., in D. Dabbs, ed.,DIAGNOSTIC IMMUNOHISTOCHEMISTRY 3^(rd) Edition (2011, Saunders,Philadelphia), CD11c is a type I transmembrane protein that is expressedon monocytes, granulocytes, a subset of B cells, dendritic cells, andmacrophages. Internalization of the inventive compositions into any ofthese cell types is expected to result in reduced inflammation andconsequent alleviation of symptoms of inflammation.

Conditions for Which Mutant Enterotoxins Can Be Used asAnti-Inflammatory Agents

It is anticipated that symptoms of many inflammatory diseases andconditions can be ameliorated or treated by the inventive methods andcompositions.

As enterotoxic E. coli (ETEC″) and cholera affect the mucosal lining ofthe intestinal tract, the B subunits of their toxins are particularlygood for carrying detoxified A subunits to mucosal surfaces. Thus,embodiments of the inventive compositions and methods are particularlysuited for ameliorating inflammation in the gastrointestinal tract.Chronic inflammation can occur at various sites within thegastrointestinal tract. See, e.g., Bamford, K., FEMS Immunology &Medical Microbiology, 1999, 24(2)161-168. It is contemplated that insome embodiments, the inventive compositions and methods can be usedtransmurally to ameliorate symptoms of inflammation throughout theentire tract, while in other embodiments, they can be used to amelioratesymptoms at particular sites or in particular segments, such as thebowel or colon, depending on the form and route of administration. Forexample, compositions administered orally in liquid form would heexpected to travel, and ameliorate symptoms, along the entire tract,while compositions administered in the form of a suppository would beexpected to ameliorate symptoms in the rectum. In some embodiments, anendoscope or similar instrument can be used to deliver the inventivecompositions to an affected site within the large or the smallintestines, or both. For example, a colonoscope can be used to deliveran inventive composition to the junction of the ileum and the colon.

In some embodiments, the gastrointestinal inflammation to he amelioratedis inflammatory bowel disease (sometimes referred to herein as “IBD”).According to the CDC, in 2015, an estimated 1.3% of U.S. adults, or over3 million individuals, had been diagnosed with IBD, a 50% increase fromthe number with IBD in 1999. See also, Dahlhamer, et al., MMWR MorbMortal Wkly Rep., 2016, 65(42):1166-1169.

Two major forms of IBD are Crohn's disease (sometimes referred to hereinas “CD”) and ulcerative colitis (sometimes referred to herein as “UC”).In some cases, it cannot be determined if a patient's IBD is CD or UC.In such cases, the patient may be diagnosed with indeterminate colitis.According to the Crohn's and Colitis Foundation of America (“CCF”), CDcan affect any portion of the gastrointestinal tract, but most commonlyaffects the junction of the ileum and the colon mentioned above. The CCFfurther states that inflammation due to CD can occur in patches andextend through the entire thickness of the intestinal wall. In contrast,the CCF states that UC occurs only in the colon and rectum andinflammation affects only the inner lining of the tract. Both diseasescause abdominal pain, diarrhea, and a feeling of urgency to empty thebowels, and can cause rectal bleeding. CD can cause fistulas andstrictures in the intestines. UC and, less commonly, CD, can cause toxicmegacolon, in which severe inflammation causes the colon to enlarge,which can lead to nerve and muscle damage and almost complete paralysisof the affected portion. Both UC and CD can also cause the bowel toperforate.

Both CD and UC have been extensively studied. Baumgart and Sandborn, TheLancet, 2012, 380(9853):1590-1605, provides a review of the etiology,diagnosis, and treatment of CD. See also, Hart an Ng, Medicine, 2015,43(5):282-290. UC is reviewed in, e.g., Ho et al., Medicine, 2015,43(5):276-281. UC also occurs in children; pediatric UC is discussed in,for example, Turner, Inflam. Bowel D is, 2011, 17(1):440-49. Extensiveinformation regarding UC and CD are also available in standard texts,such as Friedman and Blumberg, “Inflammatory Bowel Disease,” in Jamesonet eds., HARRISON'S PRINCIPLES OF INTERNAL MEDICINE, 20^(th) Ed. (McGrawHill Education, New York, 2018).

In animal studies underlying the present disclosure, colitis scoring wasconducted as described in Chassaing, et al., Curr Protoc Immunol, 2014,104(1): 15.25.1-15.25.14. There are a number of indices for grading UCactivity and severity in humans, which use criteria such as histology,inflammation, and endoscopic examination. D'Haens et al.,Gastroenterology, 2007, 132:763-786, present a review of a number ofactivity indices and efficacy end points for clinical trials for UC inadults. The reference reviews ten indexes for measuring the severity ofUC (e.g., the Truelove and Witts Severity Index, the Powell-Tuck Index,the Seo Index), and nine endoscopic measures of disease activity (e.g.,the Truelove and Witts Sigmoidoscopic Assessment, the Baron Score, andthe Modified Baron Score). A scoring system proposed by Geboes et al.,Gut. 2000, 47:404-409, provides a scale for grading inflammation.According to Jauregui-Amezaga, et al., Jr Crohn's and Colitis, 2017,11(3):305-313, the Geboes score is one of the most commonly usedhistological scores for grading UC, but is somewhat complicated, andproposed a simplified version. Xie et al., Gastroenterology Report,2018, 6(1):38-44, compares the Ulcerative Colitis Endoscopic Index ofSeverity (UCEIS) and the Mayo Endoscopic Score (MES), both of which weredeveloped as objective methods to measure endoscopic severity, aspredictors of the need for colectomy. Travis et al., Aliment. Pharmacol.Ther., 2011, 34:113-24, review the different definitions of remission ofUC used in various trials and propose a standard definition based onclinical symptoms and endoscopy, with histopathology as a thirddimension.

While practitioners have not agreed on a universal system for measuringimprovement or remission of UC, all the indices mentioned above areintended to provide measures of improvement or worsening of thecondition and all are accepted by some portion of the medical community.For purposes of the present disclosure, it is contemplated that areduction of activity score in any of the activity indexes noted in thereferences above, or a reduction of the endoscopic activity score as setforth in any of the indexes set forth in those references indicates anamelioration of symptoms of inflammation due to UC.

In view of the reduction of T cell regulatory activity seen in studiesunderlying the present disclosure, it is believed that, in addition toIBD, embodiments of the inventive compositions and methods can be usedto reduce or ameliorate symptoms of inflammation in a number of otherconditions in which relieving symptoms of inflammation would be ofbenefit to the patient. In some embodiments, the conditions are ones inwhich other anti-inflammatory agents have been found to be helpful. Forexample, in some embodiments, the inventive compositions can beadministered to persons suffering from asthma, in addition to, or inplace of, the inhaled corticosteroids currently used as long-term asthmacontrol medications, or as a quick-relief agent to reduce airwayinflammation during severe asthma. It is contemplated that in theseuses, the inventive compositions will be administered by inhaler, usingdevices similar to those used to deliver current asthma medications.

In some embodiments, the inventive compositions and methods can be usedto relieve inflammation in joints caused by various forms ofinflammatory arthritis, such as rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, and juvenile idiopathic arthritis. Inthese embodiments, it is contemplated that the inventive compositionswill be mixed into formulations suitable for carrying the compositionsinto the skin. Creams and ointments for introducing therapeutic agentsinto the skin are well known. Other conditions for which topicalapplication is expected to be useful are dermatitis and psoriasis. Itshould be noted that a transdermal patch containing 37.5 μg E. coli LTas a vaccine was tested in clinical trials and found to shorten episodesof traveler's diarrhea and result in fewer loose stools (Frech et al.,Lancet 2008,371(9629):2019-25). A Phase 3 study of a skin patchcontaining 37.5 μg E. coli LT found that the LT was deliveredeffectively and was immunogenic. Behrens et al., The Lancet, 2014,14(4197-204, showing that LT can be delivered through the skin intoantigen presenting cells. The inventive compositions and methods arealso expected to also be useful in reducing inflammation of internalorgans, including conditions including nephritis, pancreatitis, andliver inflammation, as well as systemic inflammatory diseases, such aslupus. In these embodiments, the compositions are conveniently deliveredto the organs by intravenous (IV) infusion.

Formulations

Formulations of the inventive compositions will depend in part on thesite of the inflammatory condition whose symptoms are to be treated orameliorated, and the contemplated route of administration. In the murinestudies reported in the Examples, detoxified LT E112K administered indrinking water was shown to be effective in several different murinemodels of inflammatory bowel disease. Based on these studies,formulations for reducing inflammation in IBD, including Crohn's diseaseand ulcerative colitis, could be as simple as providing a subject inneed thereof water containing a composition of a detoxified A subunitand a carrier of choice. The water preferably contains a smallpercentage of NaCl or another pharmacologically acceptable salt tomaintain conformational stability of the proteins and to maintain theproteins in solution. Preferably, the salt is present at 0.1M. In someformulations, the detoxified A subunit and carrier of choice may beprovided in lyophilized form, and reconstituted in water prior to beingtaken orally by the subject. These formulations may be given orally or,for example, may be administered through an endoscope to a desired sitein the gastrointestinal tract.

For oral administration, FDA-approved flavoring ingredients andsweeteners that are compatible with the detoxified A subunit and carrierof choice may be added. It is unlikely that any particular generallyused flavoring ingredients or sweeteners are not compatible withadministration of the detoxified A subunit and carrier of choice, butany particular combination can be readily tested by performing twoparallel studies following the protocol described in Example 5, below,or in Example 6,below, one in which the detoxified A subunit and carrierof choice are provided in water and one in which the detoxified Asubunit and carrier of choice are provided in water with the combinationof flavoring ingredient and sweetener to be tested, and comparing theresults. If the addition of the flavoring ingredient and sweetener tothe water with the detoxified A subunit and carrier of choice results inreducing the benefit to the mice seen by use of the detoxified A subunitand carrier of choice in water alone, that combination of flavoringagent and sweetener is not a compatible combination with the detoxifiedA subunit and carrier of choice. In similar formulations, thecomposition can be provided as syrups or suspensions.

In some embodiments, the detoxified A subunit and carrier of choice maybe administered in a suitable oral dosage form, such as a pill, capsule,tablet, lozenge, pastille, pellet, medicated chewing gum, powder,solution, suspension, wafer, or syrup. Making such oral dosage forms iswell known in the art, and it is expected that persons of skill arefamiliar with the considerable literature and guidance that exists, asexemplified by texts such as Gennaro, A., REMINGTON'S PHARMACEUTICALSCIENCES, 18^(th) Ed., (1990), Rowe, Shesky and Quinn, eds. HANDBOOK OFPHARMACEUTICAL EXCIPIENTS, 6^(th) Ed. (Pharmaceutical Press, London,2009) and L. Allen, ed., REMINGTON: THE SCIENCE AND PRACTICE OFPHARMACY, vols I and II, 22^(nd) Ed. (Pharmaceutical Press,Philadelphia, 2012).

In addition to the detoxified A subunit and carrier of choice, theformulations may include one or more pharmaceutically acceptableexcipients, stabilizers, binders, lubricants, tillers, buffers,antioxidants, such as ascorbic acid, preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; orbenzalkonium chloride), monosaccharides, disaccharides, and other sugarsor carbohydrates, including glucose, mannose, sucrose, mannitol,trehalose or sorbitol, low molecular weight polypeptides, proteins, suchas serum albumin or gelatin, hydrophilic polymers such aspolyvinylpyrrolidone, chelating agents such as EDTA, and salt-formingcounter-ions such as sodium. In some embodiments, the pills, capsules,or tablets may be formulated to be taken bucally (against the cheek) orsublingually (under the tongue) or to be orally disintegrating. In someembodiments, the compositions may be included in a film that dissolvesand releases the compositions when administered buccally orsublingually, or in a spray that is so administered to the mouth or tothe nasal cavity. In some embodiments, the pill, capsules, or tabletsmay be formulated as modified release dosage forms, including delayed,extended, sustained, pulsed, or fast release forms.

Formulations intended for application to the skin are typically in anointment base of a thick oil in a 80% oil to 20% water mixture with ahigh viscosity. A number of bases, such as beeswax, hydrocarbon bases,such as ceresine, and vegetable oil bases are known and can be selecteddepending on the particular properties of penetrability, stability,solvent property and release of medicament desired by the practitioner.The inventive compositions and methods can also administered topicallyin the form of gels or in transdermal patches, such as those used in theclinical trials testing LT as a vaccine for enterotoxic E. colidiscussed in the preceding section.

Dosing and Administration

For purposes of the inventive methods, an “effective amount” or a“therapeutically effective amount” of a composition comprising adetoxified LT A subunit refers to an amount that, alone or incombination with further doses, produces the desired response, e.g.,alleviation or elimination of inflammation. This may involve decreasingthe amount of inflammation only temporarily or only briefly, so long asit is measurable. This can be measured by standard methods, such as theUC activity scores and endoscopy score indices discussed in a precedingsection.

The “therapeutically effective amount” will depend on the particularcondition being treated, the severity of the condition, patientcharacteristic such as age, physical condition, size, gender, andweight, the duration of treatment, the nature of concurrent therapy, ifany, the specific route of administration, and similar factors. Thesefactors are well known to treating physicians, who are trained to makedecisions regarding treatment regimens and dosages based on these andsimilar criteria. It is generally preferred that the patient beadministered the highest safe dose according to clinical judgment, butit is understood, as with many therapeutics, that the patient may wishto take a lower dose for any of a variety of reasons. It is alsounderstood that the practitioner's decision about how much to prescribeto a particular patient will be guided by information obtained duringclinical trials of the compositions.

In some embodiments, the practitioner may evaluate a patient for theseverity of UC before commencing administration of an induction dose andthen reevaluate the patient following the induction dose to determine ifthere has been a desired clinical effect, as shown by any of endoscopicimprovement, endoscopic remission, clinical response, an improvement inscoring under the practitioner's choice of disease activity index orendoscopic index, rectal bleeding subscore, endoscopic subscore,histologic improvement, and combinations thereof.

For topical administration or administration by injection, it iscontemplated that the dose range will be from about 1 μg (with “about”here meaning±0.1 μg) to about 5 mg (with “about” here meaning±0.2 mg.All dosages stated herein refer to the amount of detoxified A subunitand carrier.) In some embodiments in which the amount of detoxified Asubunit and carrier administered is 100 μg or less, the compositionadministered does not also contain an exogenous antigen to which thepractitioner wishes to raise an immune response. In some embodiments inwhich the amount of detoxified A subunit and carrier administered is 75μg or less, the composition administered does not also contain anexogenous antigen to which the practitioner wishes to raise an immuneresponse. In some embodiments in which the amount of detoxified Asubunit and carrier administered is 50 μg or less, the compositionadministered does not also contain an exogenous antigen to which thepractitioner wishes to raise an immune response.

For oral administration, it is contemplated that the dose range will befrom about 100 μg (with “about” here meaning±5 μg) to about 500 mg ofdetoxified A subunit and carrier (with “about” here meaning±2 mg.) Forsome conditions causing the inflammation being treated, compositionscomprising the detoxified A subunit and carrier may be administered in asingle dose intended to reduce symptoms of inflammation, while inothers, the compositions will be administered in multiple doses. In someembodiments, it is contemplated that multiple doses of compositionscomprising the detoxified. A subunit and carrier will be administered inan larger “induction dose,” followed by one or more smaller “maintenancedoses.” In these embodiments, it is contemplated that the largerinduction dose will induce an initial reduction of symptoms ofinflammation, while the following, maintenance, doses will be at a lowerdosage to keep the inflammatory symptoms in check and, preferably,wholly suppressed.

For amelioration of inflammation due to UC or CD, which present aschronic conditions, maintenance doses may be administered, for example,daily, every other day, biweekly, weekly, or every two weeks, for aperiod of months, or indefinitely. If maintenance doses alone are notkeeping symptoms of inflammation in check, one or more additionalinduction doses may be administered to reduce or bring symptoms ofinflammation under control before returning to, and continuing with,maintenance doses. For example, if the inflammatory condition is UC, andthe patient's UC activity score has regressed from one achieved after aninduction dose, the patient can administered one or more additionalinduction doses. The additional doses can be administered daily,biweekly, or weekly until clinical improvement is seen. In someembodiments, the induction doses may be continued until there is aclinical remission as determined, for example, by endoscopic examinationor the patient's subjective evaluation of quality of life, includingreduction or lack of tenesmus, whereupon the patient can be started on,or returned to, maintenance doses. It is contemplated that inductiondoses administered for UC, CD, or other IBDs, will be 1 mg to 500 mg,about 2-400 mg, about 2-300 mg, about 5-200 mg, about 5-150 mg, about5-100 mg, about 5-50 mg, or about 5-25 mg with each succeeding rangebeing more preferred than the one before it and “about” in this contextmeaning±1 mg. It is further contemplated that maintenance doses willrange from 1 mg to 300 mg, 1 mg to 200 mg, about 2-200 mg, about 2-150mg, about 2-100 mg, about 2-75 mg, about 2-50 mg, about 2-40 mg, about2-30 mg, about 2-20 mg or about 2-10 mg, with each succeeding rangebeing more preferred than the one before it and “about” in this contextmeaning±1 mg.

In the animal studies reported in the Examples, mice with differentmodels of IBD showed reduction of symptoms when administered dailydetoxified A subunit and carrier in water which contained 0.3% NaCl.Accordingly, in some embodiments, the patient may take daily doses ofdetoxified A subunit and carrier in a suitable liquid, such as water. Insome embodiments, a dose for daily administration may be 1 mg to 500 mg,1 mg to 300 mg, 1 mg to 200 mg, about 2-200 mg, about 2-150 mg, about2-100 mg, about 2-75 mg, about 2-50 mg, about 2-40 mg, about 2-30 mg,about 2-20 mg or about 2-10 mg, with each succeeding range being morepreferred than the one before it and “about” in this context meaning+1mg.

In veterinary applications, it is contemplated that the veterinarianwill make the dosing decision based on the species, size, gender, age,physical condition, and weight of the animal, the duration of treatment,the nature of concurrent therapy, if any, the specific route ofadministration, and similar factors, in determining the amount ofdetoxified A subunit and carrier to administer. The formulations willtypically be administered in the animal's drinking water. If the animalis free ranging, or has alternative water sources available and does notappear to like the taste of drinking water containing the therapeuticcomposition, the detoxified A subunit and carrier can be introduced intothe animal by other methods used to administer veterinary drugs, such asby injection, by adding it as a food additive, or by administering thecomposition as a pill.

EXAMPLES Example 1

This Example sets forth some methods and materials used in the studiesdiscussed below.

Production of the E112K Mutant

E112K was prepared by galactose-affinity chromatography as describedpreviously (Clements and. Finkelstein, Infect Immun. 1979, 24(3):760-9,Cheng et al., Vaccine, 2000, 18:38-49). Briefly, toxins were purifiedfrom E. coli expression cultures grown overnight in a 10-literfermenter. Cells were harvested by centrifugation and lysed in amicrofluidizer model M-110L (Miocrofluidics, Newton, Mass.). The celllysates were dialyzed overnight in TEAN (0.05 M Tris, 0.001 M EDTA,0.003 M NaN₃, 0.2 M NaCl, pH 7.5), clarified by centrifugation, andsubjected to chromatography on separate immobilized D-galactose columns(Pierce, Rockford, Ill.). Toxins were eluted with 0.3 M galactose inTEAN and passed through an endotoxin removal column (Pierce, Rockford,Ill.). The composition and purity of each protein was confirmed bySDS-PAGE (FIG. 1 and FIG. 9) and Limulus Amebocyte Lysate assay (LonzaInc, Walkersville, Md.). The endotoxin content of the final products was<1 EU/mg. For final storage, 1 mg/ml solutions of protein were vialed inglass vials, frozen overnight, and then lyophilized.

Example 2

This Example shows that E112K prevents basal cytokine secretion andintracellular cAMP accumulation in intestinal cells.

As shown in FIGS. 2A-C, the E112K mutant is non-toxic, maintaining cellviability without inducing intestinal secretion. Confluent 6-well platesof epithelial cells or macrophages that were cultured with media aloneor combined with E112K doses for 24 hours in 2 mls total volume. FIG. 2Apresents Brightfield images of these culture wells. FIG. 2B showsviability staining results post-imaging by trypsinizing cells, washing,and staining for cytometric analyses in comparison with cells killed byheating to 95° C. for 3 mn. Data is shown as % dead cells. In a furtherstudy, BALB/c mice were administered E112K or LT by oral gavage. After 3hours, the mice were euthanized and the gut/carcass weight ratiosestablished. The results are shown in the graph presented as FIG. 2C.(Significance by one-way ANOVA analysis with Tukey's multiple comparisonpost-hoc test is shown as *P≤0.05, **P≤0.01 and ***P≤0.001 versuscontrols.)

It has been established for many years that LT induces cAMP accumulationthrough binding to host cell ARF to initiate ADP-ribosylation of Gsα,leading to irreversible activation of adenylate cyclase and increasedproduction of intracellular cAMP (which ultimately leads to secretorydiarrhea). E112K maintains the strong cellular binding andinternalization properties of its parent molecule. Its A- and B-subunitscan be detected inside human intestinal epithelial cells for up to sevendays after in vitro treatment (FIG. 3A). In polarized epithelial cellmonolayers, E112K lacks the ability to induce cAMP accumulation andsubsequent ion secretion compared to the more toxic LT (FIGS. 3B, 3D).In the same culture system, our data indicates that while inflammatoryIL-6 cytokine responses are slightly diminished, there is no majorimpact observed any observable changes on transwell epithelial monolayerintegrity (FIGS. 3C, 3D). Similarly, in non-polarized monolayers ofepithelial cells, E112K induces a 1000×-fold less cAMP productioncompared with 0.001 μg LT after 3 h treatments (in the presence ofphosphodiesterases) and is consistent between protein lots of E112K evenat 10 μg treatment or 10,000× excess protein compared with LT (FIG. 10).Intriguingly, pre-treatment of cells with E112K prevents LT-mediatedcAMP accumulation (FIG. 3E). Thus, there is no evidence that E112Kdisrupts normal intestinal function beyond acting as ananti-inflammatory agent. These results further demonstrate a surprisingproperty of E112K in prevention of intracellular signaling. Withoutwishing to be bound by theory, we surmise that E112K sequesters hostcell ARF, which then limits molecular signaling pathways resulting indecrease cytokine production and intracellular cAMP accumulation uponexternal cellular stimulation (FIG. 11).

Example 3

This Example shows that E112K inhibits intracellular cAMP,ADP-ribosylation, and activation of dendritic cells.

One of the consequences of LT intoxication of cells is ADP-ribosylationof host cell proteins and accumulation of cAMP. in dendritic cells, thisleads to activation and upregulation of costimulatory surface markers,such as CD80 and CD86. Dendritic cells are key initiators of adaptiveimmune responses, linking the traditional innate and adaptive arms ofthe immune system. E112K is rapidly internalized and its A- andB-subunits detected in treated dendritic cells (FIG. 4A). Unlike LT,E112K will prevent cAMP accumulation in stimulated dendritic cells (FIG.4B). Furthermore, pretreatment of dendritic cells with E112K inhibitsintracellular ADP-ribosylation of host cell proteins by LT andLT-mediated activation (FIGS. 4B, 4C). Pre-treatment of dendritic cellswith E112K, prior to LPS-stimulation also results in decreased dendriticcell activation. These changes are not seen with LTB treatment, whichonce again strongly indicates a key role for E112K's inactivatedA-subunit in its immunosuppressive properties. These results confirm thesurprising property of E112K in preventing intracellular signaling andcellular activation.

Example 4

This Example shows that E112K inhibits adaptive immunity.

For decades, LT has been known as a potent mucosal and parenteraladjuvant, inducing immune responses to admixed antigens. Older studiesalso clearly determined that E112K and related mutants with mutations inthe A-subunit (e.g., E110D, S61F) that prevent ADP-ribosylation activityare not oral adjuvants (Lobet et al, Infect Immun. 1991, 59(9):2870-9,Cheng et al., Vaccine, 2000, 18:38-49). Similarly, in our studies,increasing doses of E112K admixed with tetanus toxoid result in aprogressive decline in antigen-specific antibody responses (FIG. 5).While isolated B-subunit (LTB) is also not a good adjuvant, it does notlead to the progressive decrease in adaptive immunity seen with E112K.Thus, unlike other members of the LT protein family, E112K acts uniquelyon the immune system and can inhibit antigen-specific responses tohighly immunogenic antigens. This may be an ideal property duringautoimmune inflammatory diseases, such as antigen specific responses tospecific commensal bacterial that have been reported for Crohn's diseaseand ulcerative colitis.

Example 5

This Example shows that E112K improves inflammatory bowel diseasepathologies in dextran sulphate sodium (DSS) colitis mouse models. Theclassic DSS chemical injury model of chronic, ulcerative colitis wasinduced in BALB/c mice by successive waves of 5-day drinking watertreatment with 4% DSS, followed by 7-days of water. After the 2nd (day17) or 3rd (day 29) DSS treatment, some mice were treated with 50 μgE112K by oral gavage. The final weight change (day 36, % originalweight) or colon length (day 38, mm) were evaluated for all groups. Theresults are presented in FIG. 6A. FIG. 6B presents H&E pictures ofswiss-rolled, processed colonic mouse intestines (from day 38 collectedsamples).

The intestines show some moderation of DSS-mediated cellularinfiltration (black asterisk) with E112K therapy.

Example 6

This Example shows that E112K is a sate, efficacious therapy forinflammatory bowel disease pathology in the IL-10-/- colitis mousemodel.

FIG. 7A shows that the weight gain over time after 200 μg E112K by oralgavage in IL-10-/- female and male mice is similar to that of naïve(control) mice. The chronic colitis model was induced in IL-10-/- micewith 7 day exposure to 200 ppm piroxicam at 6-weeks of age. Piroxicamdiet (in food gel, formulated food, or powder food) induces colitispathology at week 10. Colon histology scores were tallied usingcomposite scoring of 4 colonic sections for type and extent of mucosaland inflammatory changes by a pathologist blinded to the animaltreatments. As shown in FIG. 7B, 200 μg E112K by oral gavage (“O”) didnot induce any intestinal pathology. FIG. 7C presents examples ofH&E-stained colons from piroxicam-treated animals exhibiting typicalpathology, including hyperplasia (top photo, asterisk) and ulceration ofintestinal wall (bottom photo, asterisk). The chronic colitis model wasinduced in IL-10-/- mice with 7 day exposure to 200 ppm piroxicam at6-weeks of age. FIG. 7D presents the colon histology score of controlmice and of mice subjected to piroxicam exposure followed after thatexposure by weekly E112K treatment by either intraperitoneal (“IP”)injection or oral gavage (“O”) for three weeks. In a separateexperiment, groups of mice (n=5-6) were exposed to piroxicam and theneither untreated or given 100 μg E112K by oral gavage weekly for threeweeks. On week 10, mice were fed 4 kD FITC-Dextran at 60 mg/1.00 g bodyweight by oral gavage 3 h prior to euthanasia. Serum was collected andthen analyzed for levels of fluorescent protein after 3 h oral gavagewith at week 10. The results are shown in FIG. 7E. Significance is shownas *P≤0.05, **P≤0.01 and ***P≤0.001.

Example 7

This Example discusses and extends the studies reported in Example 6.

As indicated in FIG. 6, oral E112K ameliorates symptoms of a chemicallyinduced model of inflammatory bowel disease (via. DSS), including weightloss, colon shortening and epithelial cell hyperplasia and mucosalinflammation. In the IL-10-/- model of chronic colitis, up to 200 μgoral E112K therapy alone does not impact weight loss or development ofIBD pathology (FIGS. 7A, 7B).

However, in piroxicam-treated mice that have observable histopathology,including architectural distortion like hyperplasia of the intestinalmucosa, inflammation, and ulceration (FIG. 7C, asterisks), weeklytreatment with 100 μg E112K for three weeks by oral gavage improvedhistology scores and reduced intestinal permeability (FIGS. 7D, 7E).These changes were similar to 10 μg E112K orally but greater than just 1or 10 μg of E112K parenterally in reducing intestinal pathology (FIG.7D).

Finally, we tested E112K for efficacy in a third model of inflammatorybowel disease, the T-cell transfer model of colitis. In this model,CD4+CD45RB-hi T-cells are transferred into Rag1-/- mice, causingunchecked intestinal inflammation and wasting (or weight loss) by 20weeks. The protocol is presented graphically in FIG. 8A. Mice thatreceived 100 μg. E112K by oral gavage on weeks 13, 16, and 18 (“oE112K”) exhibited improved pathology scores (FIG. 8B), showed noabnormal weight loss (FIG. 8C), and showed normal stool consistency(FIG. 8D).

Thus, E112K was shown to be an effective therapy for IBD in threedifferent mouse models of colitis.

Example 8

This Example shows that the exemplar detoxified A subunit-carrier E112Kimproved intestinal permeability as soon as 24 hours post-treatment. Thechronic colitis model was induced in IL-10-/- mice with 7 day exposureto 200 ppm piroxicam at 6-weeks of age. IL-10-/- mice were exposed topiroxicam for 7 days in rodent chow and then 4 days later either leftuntreated or treated with E112K in drinking water. Intestinalpermeability was tested 1-day after treatment by analyzing serum forfluorescent protein after 3 h oral gavage with 4 kD FITC-Dextran. Theresults are shown in FIG. 11. Significance is shown as *P≤0.05.

Example 9

This Example shows that the exemplar detoxified A subunit-carrier E112Kimproved disease activity index in DSS acute model of colitis inimmunodeficient mice. Rag1-/- mice that lack functional adaptive immunesystem were exposed to 3% DSS in drinking water for 7 days. A group ofthese DSS mice were treated with oral E112K on day 5. Weight changes anddisease activity index (evaluated by weight loss, fecal consistency, andblood in feces) were evaluated in animals at day 8. The results areshown in graph form in FIG. 12. As shown in FIG. 12, E112K treatmentimproved the disease activity index, but not weight loss, indicatingthat at least some of E112K immunomodulatory effects are T-cellindependent.

Example 10

1×10⁶ bone-marrow derived mouse dendritic cells were treated intriplicate for 48 h with 0.1 μg/ml test proteins before analysis of CD80and CD86 co-stimulatory marker levels on CD11c+ gated cells. Assay wasperformed three independent times. FIG. 13A presents representative dotblots and mean fluorescent intensity levels (MFI) from one experiment.Th2-promoting cytokines and Th1/Th17-promoting cytokines were assayedfrom culture supernatants of 1×10⁶ CD11c+ purified DC cultured with 0.1μg/ml test proteins in triplicate for 5 days. As shown in FIGS. 13B andC, ETEC LT B subunit (“LTB”) partially activated dendritic cells,similar to holotoxin (“LT”) and mutated AB₅ proteins that act asadjuvants, mLT (LT-R192G), and dmLT (LT-R192G/L211A), includingincreased expression of co-stimulatory marker CD86 and secretion ofIL-12p70, RANTES, and IL-6 cytokines, while E112K did not induce theexpression or secretion of these or other cytokines. The expression ofIL-6 induced by E112K, for example, was similar to that of the controlgroup, while high levels of IL-6 were induced by LT, LTB, and the twomutated holotoxins. Similar cytokine profiles can be seen for the otherTh1-promoting cytokines assayed and for the other Th17-promotingcytokines assayed.

Example 11

This Example discusses a possible mechanism of action for detoxified LTmutants such as E112K.

Without wishing to be bound by theory, it is believed that the intactdetoxified LT mutant E112K binds to GM1 surface receptors on cells suchas epithelial cells through B-subunit binding. It is further believedthat the binding results in release of the E112K A1-subunit into thecytosol, that it is the A1-subunit that interferes with function ofADP-ribosylation factor (ARF) in the host cell, and that it is theinterference of the A1-subunit with ARF activity that inhibits ongoingvesicular trafficking and innate signaling (e.g., IL-6 cytokinesecretion) and decreases cAMP levels over time, ultimately dampeningoverall levels of inflammation. Thus, it is believed that the A1-subunitinterference with ARF activity suppresses the release of inflammatorycytokines, suppresses pro-inflammatory signaling responses, andsuppresses inflammation, as shown in cartoon form in FIG. 13. Since itis believed that the role of the B-subunit is to act as a carrier tocarry the A1-subunit into the cell and allow it to translocate from theGolgi apparatus to the cytosol, it is believed that other carriersenabling cellular targeting, cellular entry, or both, can be used ascarriers of the A1-subunit and act to reduce inflammation in subjects towhich such carrier-detoxified LT A1-subunits are administered.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A composition comprising a unit dose of: (a) (i) an E. coli heat labile enterotoxin (“LT”) non-toxic A subunit which inhibits ADP-ribosylation in a cell pretreated with said non-toxic A subunit when said cell is then contacted with E. coli LT holotoxin, or (ii) a LT non-toxic A1 subunit which inhibits ADP-ribosylation in a cell pretreated with said non-toxic A subunit when said cell is then contacted with E. coli LT holotoxin, or, (iii) a combination of said non-toxic A subunit and of said non-toxic A1 subunit, and (b) a carrier which causes internalization of said non-toxic A subunit or non-toxic A1 subunit, or combination thereof, into cells.
 2. The composition of claim 1, further wherein said non-toxic A subunit or said non-toxic A1 subunit, or said combination of said non-toxic A subunit and said non-toxic A1 subunit does not induce intracellular cAMP accumulation in an epithelial cell.
 3. The composition of claim 1, further wherein said non-toxic A subunit or said non-toxic A1 subunit, or said combination of said non-toxic A subunit and said non-toxic A1 subunit does not activate expression and secretion of IL-6 in murine dendritic cells contacted with said non-toxic A subunit or said non-toxic A1 subunit, or said combination of said non-toxic A subunit and said non-toxic A1 subunit in vitro in a medium that supports growth of said murine dendritic cells, compared to said cells contacted with LT B subunit.
 4. The composition of claim 1, wherein said carrier which causes internalization of said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a LT B subunit and said unit dose is from 0.5 mg or more of non-toxic A subunit, non-toxic A1 subunit, or combination thereof and said B subunit carrier.
 5. The composition of claim 1, wherein said carrier which causes internalization of said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is cholera toxin B subunit (“CTB”) or cholera A2 subunit/B subunit (“CTA2/B”).
 6. The composition of claim 4, wherein said non-toxic A subunit, non-toxic A1 subunit, or combination thereof is chemically conjugated or recombinantly fused to said LT B subunit.
 7. The composition of claim 5, wherein said non-toxic A subunit, non-toxic A1 subunit, or combination thereof is chemically conjugated or recombinantly fused to said CTB or CTA2/B subunit.
 8. The composition of claim 1, wherein said carrier which causes internalization of said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a liposome or encapsulating vesicle.
 9. (canceled)
 10. The composition of claim 1, wherein said liposome or encapsulating vesicle is a liposome, said liposome has an exterior surface, and said exterior surface has an antibody or antigen-binding fragment or derivative thereof disposed thereon.
 11. The composition of claim 10, wherein said antibody or antigen-binding fragment or derivative binds CD11c.
 12. The composition of claim 1, wherein said carrier which causes internalization of said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a β-glucan. 13-15. (canceled)
 16. The composition of claim 1, wherein said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, is a combination of non-toxic A subunit and non-toxic A1 subunit. 17-18. (canceled)
 19. The composition of claim 1, wherein said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, has a mutation at position E112, E110, S61, or R25.
 20. The composition of claim 19, wherein said mutation is selected from E112K, E112G, E112D, E110K, E110G, S61F, and R25G. 21-29. (canceled)
 30. A method of reducing symptoms of inflammation in a subject in need thereof, said method comprising administering to said subject a composition comprising a therapeutically effective amount of: (a) (i) an E. coli heat labile enterotoxin (“LT”) non-toxic A subunit which inhibits ADP-ribosylation in a cell pretreated with said non-toxic A subunit when said cell is then contacted with E. coli LT holotoxin, (ii) a LT non-toxic A1 subunit which inhibits ADP-ribosylation in a cell pretreated with said non-toxic A subunit when said cell is then contacted with E. coli LT holotoxin, or, (iii) a combination of said non-toxic A subunit and said non-toxic A1 subunit, and, (b) a carrier which causes internalization of said non-toxic A subunit or non-toxic A1 subunit, or combination thereof, into cells.
 31. The method of claim 30, further wherein said non-toxic A subunit or said non-toxic A1 subunit, or said combination of said non-toxic A subunit and said non-toxic A1 subunit does not induce intracellular cAMP accumulation in an epithelial cell.
 32. The method of claim 30, further wherein said non-toxic A subunit or said non-toxic A1 subunit, or said combination of said non-toxic A subunit and said non-toxic A1 subunit does not activate expression and secretion of IL-6 in murine dendritic cells contacted with said non-toxic A subunit or said non-toxic A1 subunit, or said combination of said non-toxic A subunit and said non-toxic A1 subunit in vitro in a medium that supports growth of said murine dendritic cells, compared to said cells when contacted with LT B subunit
 33. The method of claim 30, wherein said carrier which causes internalization of said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a LT B subunit or a cholera toxin B subunit (“CTB”) or cholera A2 domain and B subunit (“CTA2/B”), optionally wherein said non-toxic A subunit, non-toxic A1 subunit, or combination thereof is chemically conjugated or recombinantly fused to said LT B subunit or to said CTB or said CTA2/B subunit, respectively. 34-36. (canceled)
 37. The method of claim 30, wherein said carrier which causes internalization of said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a liposome or encapsulated vesicle.
 38. (canceled)
 39. The method of claim 38, wherein said carrier is a liposome and said lipsome is targeted to a cell antigen by an antibody or antigen-binding fragment or derivative thereof.
 40. The method of claim 30, wherein said carrier which causes internalization of said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a β-glucan. 41-47. (canceled)
 48. The method of claim 30, wherein said non-toxic A subunit, non-toxic A1 subunit, or combination thereof has a mutation in said A subunit, said A1 subunit, or both, selected from the group consisting of E112K, E112G, E112D, E110K, E100G, S61F, or R25G. 49-58. (canceled)
 59. The method of claim 30, wherein said therapeutically effective amount is administered as an induction dose followed by one or more maintenance doses.
 60. (canceled)
 61. The method of claim 30, wherein said therapeutically effective amount of said composition is from 500 μg to 500 mg. 62-68. (canceled)
 69. The method of claim 30, wherein said inflammation is inflammatory bowel disease, optionally wherein said inflammatory bowel disease is ulcerative colitis or Crohn's disease. 70-71. (canceled)
 72. The method of claim 30, wherein said inflammation is of the skin, optionally wherein said inflammation of the skin is psoriasis or dermatitis.
 73. (canceled)
 74. The method of claim 30, wherein said inflammation is (a) a form of inflammatory arthritis selected from rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or juvenile idiopathic arthritis, or (b) inflammation of an internal organ other than the small or large intestine, bowel, or colon. 75-77. (canceled) 